Volume 48, Issue 4, Pages 521-531 (November 2012) Stress-Induced Alternative Splicing Provides a Mechanism for the Regulation of MicroRNA Processing in Arabidopsis thaliana  Kang Yan, Peng Liu, Chang-Ai Wu, Guo-Dong Yang, Rui Xu, Qian-Huan Guo, Jin-Guang Huang, Cheng-Chao Zheng  Molecular Cell  Volume 48, Issue 4, Pages 521-531 (November 2012) DOI: 10.1016/j.molcel.2012.08.032 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 48, 521-531DOI: (10.1016/j.molcel.2012.08.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Genomic Structure and Expression Patterns of Intronic miRNA400 and Its Host Gene in Arabidopsis (A) Schematic diagrams of the structure of Arabidopsis gene At1g32583 containing intronic miR400. UTRs are shown in blue, exons in orange, and introns in thick lines. The detailed imformation of intron 1 containing primary miR400 is indicated. Gray boxes represent ordinary intron region, the red box represents the cryptic AS region, and stem loop represents miR400 precursor. Numbers above each region indicate the length of fragments. (B and C) Tissue patterns of MIR400 and its host gene transcript accumulation. Real-time RT-PCR quantifications were normalized to the expression of Tubulin. Error bars represent SE for three independent experiments. (D) Tissue patterns of mature miR400 accumulation. Real-time RT-PCR quantifications were normalized to the expression of U6 RNA. Error bars represent SE for three independent experiments. R, root; RL, rosette leaf; CL, cauline leaf; St, stem; F, flower; Si, silique. Total RNA was isolated from various tissues of 4-week-old wild-type plants grown under long-day growth conditions. See also Figure S1. Molecular Cell 2012 48, 521-531DOI: (10.1016/j.molcel.2012.08.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Regulation of miR400 and Host Gene Expression by Heat Treatment (A) GUS staining in 3-week-old transgenic seedlings on MS-agar medium that were exposed to heat treatment for 12 hr or untreatment for 12 hr (control). (B) Quantification of GUS activity in 3-week-old transgenic seedlings on MS-agar medium that were exposed to heat treatment for 12 hr. The results are means ± SD of GUS activities from three independent experiments. (C) RNA gel blot analysis of regulation of mature miR400 by heat stress. miR171 or U6 RNA was probed as a loading control. Numbers below each lane indicate relative expression. (D) Heat stress induced MIR400 transcripts but do not affect host gene expression. Real-time RT-PCR quantifications were normalized to the expression of Tubulin. The transcript level in the controls was set at 1. Error bars represent SE for three independent experiments. (E) Detection of mature miR400 in response to heat stress by real-time RT-PCR. Quantifications were normalized to U6 RNA. See also Figure S2. Molecular Cell 2012 48, 521-531DOI: (10.1016/j.molcel.2012.08.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Alternative Splicing Event of the miR400-Containing Intron in Response to Heat Stress (A) Diagrammatic representation of the AS isoforms as deduced from sequencing products under heat treatment or untreatment (intron 1 mock, intron 1 heat, UTR mock, and UTR heat). The arrow indicates the position of the RT-PCR primers which were used in RT-PCR experiments (intron 1 primers and UTR primers). Numbers beside each region indicate the length of fragment. (B) Proposed splicing models of intron containing the miR400 hairpin. Different events (1–3) were kept separate by the curve. Event 1, Unspliced pre-mRNA hosting the intron; event 2, the intron spliced out and the primary miR400 released from the pre-mRNA; event 3, triggered by heat stress, part of intron containing miR400 hairpin retained instead. Events 1 and 2 occurred in any conditions, and event 3 only occurred in response to heat stress. (C) Heat stress triggered the AS event specially. RT-PCR primer locations were shown in (A). (D and E) RT-PCR analysis of temperature and time course of AS in response to heat stress. Tubulin was used as the loading control. A no-RT control (−) was run for each RNA sample to ensure that no DNA contamination was present. (F) qRT-PCR analysis of the nascent or partially spliced transcripts containing primary miR400. Different RT primers were used to distinguish these transcripts, and error bars represent SE for three independent experiments. See also Figure S3. Molecular Cell 2012 48, 521-531DOI: (10.1016/j.molcel.2012.08.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 miR400 Is Mainly Regulated by AS under Heat Stress (A) Diagram of miR400-AS expression constructs. UTRs are shown in blue, exons in orange, ordinary introns in thick lines, the intron containing primary miR400 in gray and MIR400 as black box with “M.” The red box represents the cryptic AS intron region. All constructs were driven by the constitutive CaMV 35S promoter. (B) Coexpression of various combinations miR400-AS expression constructs and miR397 control construct in N. benthamiana. miR397 was used as an internal reference. Real-time RT-PCR quantifications were normalized to the expression of miR397. Error bars represent SE for three independent experiments. (C) Detection of mature miR400 in transgenic Arabidopsis lines with corresponding miR400-AS expression constructs by real-time RT-PCR. Quantifications were normalized to the expression of U6 RNA. Error bars represent SE for three independent experiments. (D) RNA gel blot analysis of miR400 in transgenic plant lines with corresponding miR400-AS expression constructs. miR171 and U6 RNA was probed as a loading control. Numbers below each lane indicate relative expression. Molecular Cell 2012 48, 521-531DOI: (10.1016/j.molcel.2012.08.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Mutations of Splicing Signals Inhibited Normal Processing of miR400 (A) Sequence analysis of the miR400-containing intron in the 5′UTR of host gene. Intron 1, the underlined letters; cryptic AS region, small letters with green shadow; branch site and associated polypyrimidine tract, letters in box; primary miR400 transcirpt, capital letters; mature miR400, capital letters with blue shadow. Splicing signals are indicated by arrows. (B) Original splicing signals (blue) were mutated (red) at indicated positions to generate constructs that lack the consensus sequences. 5′ SS, mutations in 5′ donor site; 5′ AS, mutations in 5′ heat-related AS site; BP-1, mutations in conserved “A” site of the predicted BPs; BP-2, mutations to another consensus BPs; BP-3, mutations in pyrimidines of the polypyrimidine tract besides the BPs. Coexpression of various combinations miR400 mutation constructs and miR397 control construct in N. benthamiana. miR397 was used as an internal reference. MIR400 - hostgene construct was used as original vector. qRT-PCR quantifications were normalized to the expression of miR397. Error bars represent SE for three independent experiments. Molecular Cell 2012 48, 521-531DOI: (10.1016/j.molcel.2012.08.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Heat-Sensitive Phenotypes of 35S::MIR400 Plants (A and B) Germination of seeds after heating to 45°C for 220 min and scoring 1 week after heat treatment. (C) Hypocotyl elongation of 2.5-day-old old seedlings and 2.5 day recovery following a 38°C pretreatment, 120 min at room temperature, and 180 min heat stress at 45°C. (D) Root elongation 5 days after heat stress for plants heated to 45°C for 180 min after pretreatment; plants were heated 4 days after germination. All other lines were significantly different from the wild-type (Student's t test, p < 0.001). Error bars in (A), (C), and (D) represent the SD over five replicate experiments, each containing at least 20 plants of each line. See also Figure S4. Molecular Cell 2012 48, 521-531DOI: (10.1016/j.molcel.2012.08.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 A Regulatory Model of Intronic miR400 Phased Processing and Splicing Signal Role in a Heat-Stress-Induced and AS-Involved Regulatory Loop In constitutive conditions, intron 1 splices out from nascent pre-mRNA by spliceosome and primary miR400 transcripts release which in turn give rise to mature miR400 by Microprocessor. The mRNA of host gene undergoes splicing, and then translation into a protein. In heat stress condition, AS event occurs in intron 1 and another isoform of host gene appears. Fragment (100 bp) contains the original branch site excised (the red box represents the cryptic AS intron region), which makes the rest an unrecognized 206 bp intron including the miR400 hairpin retain in the 5′UTR of host gene. Notably, primary miR400 transcripts do not splice out which hardly process into maturation by Microprocessor. The heat-stress-related AS led to decrease of mature miR400, but do not affect the host gene expression. Molecular Cell 2012 48, 521-531DOI: (10.1016/j.molcel.2012.08.032) Copyright © 2012 Elsevier Inc. Terms and Conditions